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SAM 122.05

 Page 1 of 13

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 122

Supplemental Assay Method for the Titration of Porcine Rotavirus Antibody

(Constant Virus-Varying Serum Method)

Date: October 21, 2014 Approved, Pending Standard Requirement

Number: SAM 122.05

Supersedes: SAM 122.04, June 28, 2011

Standard Requirement:

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 10Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 16Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 16Dec14

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for the Titration of Porcine Rotavirus Antibody

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

Appendix
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Supplemental Assay Method for the Titration of Porcine Rotavirus Antibody

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro serum neutralization (SN) method 

which utilizes cytopathic effects (CPE) or an indirect fluorescent antibody (IFA) technique in a 

cell culture system for determining the SN antibody titer against Group A porcine rotavirus 

(PROTA). The SN assay uses a constant amount of virus to test varying dilutions of serum.

Note: For this SAM, the dilution terminology of 1:10, 1:20, etc., specifies 1 part plus 9 parts 

(liquid), 1 part plus 19 parts, etc.

2. Materials

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1 Equipment/instrumentation

2.1.1 Incubator, 36°± 2°C, 5% ± 1% CO2, high humidity (Model 3158, Forma 

Scientific Inc.)

2.1.2 Water bath

2.1.3 Microscope, inverted light (Model CK, Olympus America Inc.)

2.1.4 Microscope, ultraviolet (UV) light (Model BH2, Olympus America Inc.)

2.1.5 Vortex mixer (Vortex-2 Genie, Model G-560, Scientific Industries Inc.)

2.1.6 Micropipetters: 200-μL and 1000-μL single channel; 300-μL x 12-channel

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 PROTA Positive Control Viruses

1. Serotype 4 (Gottfried strain)

2. Serotype 5 (OSU strain)
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2.2.2 Rhesus monkey kidney cells (MA-104) free of extraneous agents as tested 

by title 9, Code of Federal Regulations (9 CFR), (available from the Center for 

Veterinary Biologics (CVB))

2.2.3 Minimum essential medium (MEM) (National Centers for Animal Health

(NCAH) Media #20030)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaHCO3)

3. Q.S. to 1000 mL with deionized water (DI); adjust pH to 6.8-6.9 with 

2N hydrochloric acid (HCl).

4. Sterilize through a 0.22-μm filter.

5. Aseptically add:

a. 10 mL L-glutamine (200 mM)

b. 50 μg/mL gentamicin sulfate

6. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 900 mL of MEM

2. Aseptically add 100 mL of gamma-irradiated fetal bovine serum 

(FBS).

3. Store at 2°- 7°C.

2.2.5 Diluent Medium

1. 100 mL MEM

2. 83.3 μL pancreatin (4XNF 10X)

3. Store at 2°- 7°C.

2.2.6 Anti-PROTA monoclonal antibody (MAb)

1. MAb against Serotype 4 (Gottfried strain)
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2. MAb against Serotype 5 (OSU strain)

2.2.7 Rabbit anti-mouse fluorescein isothiocyanate labeled conjugate (Rabbit 

Anti-mouse Conjugate)

2.2.8 0.01 M Phosphate buffered saline (PBS) (NCAH Media #30054)

1. 1.19 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with DI.

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 2.0 N 

HCl.

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.9 80% Acetone

1. 80 mL acetone

2. 20 mL DI

3. Store at room temperature.

2.2.10 Cell culture plates, 96-well

2.2.11 Polystyrene tubes, 12 x 75-mm

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in the immunological basis of SN assays, cell culture 

techniques, the principles of IFA, and aseptic technique.
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3.2 Preparation of equipment/instrumentation

3.2.1 On the day of test initiation, set a water bath at 56°± 2°C.

3.2.2 On the day of test initiation, set a water bath at 36°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 MA-104 Plates. Two days prior to test initiation, seed 96-well cell culture 

plates with MA-104 cells, in Growth Medium, at a cell count that will produce a 

monolayer after 2 days of incubation at 36°± 2°C in a CO2 incubator. These 

become the MA-104 Plates. Two sera can be tested on each MA-104 Plate. 

Growth Medium is changed if excess acidity of the medium is observed or cells 

are not confluent after incubation.

3.3.2 Stock Virus Preparation. On the day of test initiation, rapidly thaw vials of 

each PROTA Positive Control Virus in a 36°± 2°C water bath. Dilute each virus 

in Diluent Medium to contain 50-300 50% tissue culture infective dose 

(TCID50)/100 μL.

3.3.3 Virus Back Titration. On the day of test initiation, make 4 serial tenfold 

dilutions of each Stock Virus.

1. Place 900 μL of MEM into 2 sets of 4, 12 x 75-mm polystyrene tubes, 

labeled 10-1to 10-4. Label each set with the appropriate stock virus.

2. Transfer 100 μL of each Stock Virus into the appropriate 10-1tubes; 

mix by vortexing. Discard pipette tip.

3. Transfer 100 μL from the 10-1tube to the 10-2tube; mix by vortexing. 

Discard pipette tip.

4. Repeat Step 3 for the remaining tubes, transferring 100 μL

sequentially from the previous dilution to the next dilution until the 

dilution sequence is completed. 

3.3.4 Working Anti-PROTA MAb. On the day of MA-104 plate examination, if 

an IFA test is to be conducted, dilute the appropriate Anti-PROTA MAb in PBS, 

as determined for that specific MAb.
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3.3.5 Working Rabbit Anti-mouse Conjugate. On the day of MA-104 plate 

examination, if an IFA test is to be conducted, dilute the Rabbit Anti-mouse 

Conjugate in PBS, according to the manufacturer’s recommendations.

3.4 Preparation of the sample

3.4.1 On the day of test initiation, heat inactivate all Test Sera in a 56°± 2°C 

water bath for 30 ± 5 minutes.

3.4.2 Prepare serial twofold dilutions of Test Sera in 96-well cell culture plates, 

which become the Dilution Plates (see Appendix). Place each Test Serum onto 2, 

96-well cell culture plates, 1 for each PROTA serotype to be tested. Make twofold 

dilutions as follows:

1. Add 150 μL Diluent Medium to all wells in Rows B-H.

2. Add 150 μL of a Test Serum to Rows A and B. Change pipette tips. 

Mix Row B with a multichannel micropipettor (6-8 fills).

3. Transfer 150 μL from Row B to Row C. Change pipette tips. Mix Row 

C with a multichannel micropipettor (6-8 fills).

4. Continue as in Step 3 for the remaining rows. Discard 150 μL from all 

wells in Row H.

5. Add 150 μL of Stock Virus to all wells of the Dilution Plate. Tap 

plates gently to mix. 

6. Incubate for 60 ± 10 minutes at 36°± 2°C to allow for neutralization of 

virus. This is an additional twofold dilution of the Test Sera. This becomes 

the Virus-Test Sera Mixture.

4. Performance of the Test

4.1 On the day of test initiation, decant Growth Medium from the MA-104 Plates.

4.2 Add 200 μL/well Diluent Medium to the MA-104 Plates. Decant the Diluent 

Medium.

4.3 Again, add 200 μL/well Diluent Medium to the MA-104 Plates. Incubate for 60 ± 

10 minutes at 36°± 2°C. Decant the Diluent Medium.
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4.4 Inoculate 200 μL/well of each Virus-Test Sera Mixture into 5 wells/dilution of an 

MA-104 Plate, using a multichannel pipettor.

4.5 Inoculate 100 μL/well of each dilution (100to 10-4) of Virus Back TitrationDiluent Medium mixture into 5 wells of an MA-104 Plate, using a multichannel pipettor. 

Add 100 μL Diluent Medium to all wells of back titration.

4.6 Add 200 μL/well of Diluent Medium to 2 columns on each MA-104 Plate, to 

serve as uninoculated cell controls.

4.7 Incubate the MA-104 Plates for 5 days postinoculation at 36°± 2°C.

4.8 CPE counting is the primary method of determining the log10 50% tissue culture 

infective dose (TCID50).

4.8.1 Five days ± 1 day postinoculation, examine the wells with an inverted 

light microscope. The CPE of PROTA is visible as cell death in the cell 

monolayer.

4.8.2 Record the number of wells/dilution showing any characteristic CPE of 

PROTA for each Test Serum and Virus Back Titration.

4.8.3 Calculate the TCID50 of each Virus Back Titration using the SpearmanKärber method as commonly modified by Finney.

4.8.4 Calculate each endpoint of the Test Sera using the Spearman-Kärber 

method as commonly modified. The endpoints of the Test Sera are reported as SN 

titer which corresponds to the reciprocal of the highest serum dilution that 

neutralizes PROTA.

Example:

1:2 dilution of Test Sera = 5 of 5 wells CPE

1:4 dilution of Test Sera = 5 of 5 wells CPE

1:8 dilution of Test Sera = 3 of 5 wells CPE

1:16 dilution of Test Sera = 0 of 5 wells CPE
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Titer = (X - d/2 + [d • S]) where:

X = Log10 of lowest dilution (=0.3)

d = Log10 of dilution factor (=0.3)

S = Sum of proportion of CPE

5 5 3 0 13

5 + 5 + 5 + 5 = 5 = 2.6

Titer = (0.3 - 0.3/2 + [0.3 • 2.6]) = 0.93

antilog of 0.93 = 8.5

Titer of the Test Serum is 9

4.9 Certain strains of PROTA may not exhibit pronounced CPE, thus an IFA may be 

conducted to determine the titer:

4.9.1 Decant the Growth Media from the MA-104 Plates.

4.9.2 Rinse the MA-104 Plates with PBS; incubate at room temperature for 5 ± 

2 minutes. Decant the PBS.

4.9.3 Fill wells with 80% acetone.

4.9.4 Incubate at room temperature for 15 ± 5 minutes.

4.9.5 Decant the 80% acetone from the MA-104 Plates and air dry at room 

temperature.

4.9.6 Pipette 35 μL of the Working Anti-PROTA MAb into all wells. Incubate 

for 45 ± 15 minutes at room temperature.

4.9.7 Fill the wells completely with PBS; incubate at room temperature for 5 ± 2 

minutes. Decant the PBS.

4.9.8 Repeat for a total of 2 washes.

4.9.9 Gently tap the MA-104 Plates onto paper towels to remove excess 

moisture.

4.9.10 Pipette 35 μL of the Working Rabbit Anti-mouse Conjugate into all wells. 

Incubate for 40 ± 10 minutes at room temperature.
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4.9.11 Repeat Sections 4.9.7 through 4.9.9.

4.9.12 Fill wells completely with DI and decant. Allow to air dry or dry at 36°± 

2°C.

4.9.13 Examine the MA-104 Plates with a UV-light microscope at 100 to 200X 

magnification.

4.9.14 A well is considered positive if typical cytoplasmic, apple-green 

fluorescence is observed.

4.9.15 Record and calculate as in Sections 4.8.2 through 4.8.4

5. Interpretation of the Test Results

5.1 The test is invalid if CPE, fluorescence, or bacterial/fungal contamination is 

observed in any of the control wells.

5.2 For a valid assay, the Virus Back Titration must be between 50 and 300 

TCID50/100 μL. 

6. Report of Test Results

Record all test results on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, U.S. Government Printing Office, 

Washington, DC.

7.2 Conrath TB. Handbook of Microtiter Procedures. In: Clinical and Research 

Applications Laboratory. Alexandria, Virginia: Cooke Engineering Co, 1972.

7.3 Finney DJ. Statistical Method in Biological Assay. 3rd ed. London: Charles Griffin 

and Co., 1978, pp. 394-401.

7.4 Rose NR, Friedman H, and Fahey JL, eds. Neutralization Assays. In: Manual of 

Clinical Laboratory Immunology. Washington, DC, ASM, 1986.
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7.5 Parker RA, Pallansch MA. Using the virus challenge dose in the analysis of virus 

neutralization assays. Statistics in Medicine 11:1253-1262, 1992.

8. Summary of Revisions

Version .05

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .04

 The Contact information has been updated.

 References to NVSL have been changed to NCAH throughout the document.

Version .03

 The Contact has been changed from Kenneth Eernisse to Joseph Hermann.

 The term “Reference” has been changed to “Positive Control” throughout the 

document. 

Version .02

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 2.2.3.2 The amount of sodium bicarbonate (NaHCO3) has been changed from 2.2 g 

to 1.1 g.

 2.2.3.5 Penicillin, streptomycin, and amphotericin B have been removed.

 2.2.4.2 “Heat–inactivated serum” has been changed to “gamma-irradiated serum”.

 3.3.2 100-700 TCID/50 has been changed to 50-300 TCID/50.

 3.3.3.1 Two sets of 8 tubes has been changed to 2 sets of 4 tubes.

 3.3.3.5 This has been deleted.
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 4.5 The volume of back titration dilutions added to the plate has been changed to be 

compatible with test conditions.

 4.8.1 120 hours ± 12 hours has been changed to 5 days ± 1 day.



 4.9.12 “Dip the plate” has been changed to “fill wells completely with DI”.

 “Reference and Reagent Sheet” has been changed to “Reagent Data Sheet” 

throughout the document.

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C. This 

reflects the parameters established and monitored by the Rees system.
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Appendix

Transfer Plate

 1 2 3 4 5 6 7 8 9 10 11 12

A

1:2

TS1 TS1 TS1 TS1 TS1 CC CC TS2 TS2 TS2 TS2 TS2

B

1:4

C

1:8

D

1:16

E

1:32

F

1:64

G

1:128

H

1:256

TS= Test Serum

CC= Cell Control